`
`(19) 0'
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`European Patent Office
`Office européen des brevets
`
`(11)
`
`EP 1 421 938 A1
`
`(12)
`
`EUROPEAN PATENT APPLICATION
`published in accordance with Art. 158(3) EPC
`
`(43) Date Of Publication:
`26.05.2004 Bulletin 2004/22
`(21) Application number: 027588805
`
`.
`.
`22 D t
`ffl
`)
`a e o ' mg
`
`(
`
`129.08.2002
`
`(84) Designated Contracting States:
`AT BE BG CH CY CZ DE DK EE ES FI FR GB GR
`IE IT LI LU MC NL PT SE SK TR
`
`(30) Priority: 31.08.2001 JP 2001264918
`
`(71) Applicant: Maeda, Hiroshi
`Kumamoto-shi, Kumamoto 862-0909 (JP)
`
`Inventors:
`(72)
`- MAEDA, Hiroshi
`Kumamoto-shi, Kumamoto 862-0909 (JP)
`
`
`
`(51)
`
`Int CI.7: A61 K 31/409, A61 K 47/48,
`A61P 35/00, A61]: 43/00,
`007D 487/22
`
`86)
`
`(
`
`International application number:
`PCT/JP2002/008707
`
`(87)
`
`International publication number:
`WO 2003/018007 (06.03.2003 Gazette 2003/10)
`
`- SAWA, Tomohiro
`Kumamoto-shi, Kumamoto 862-0918 (JP)
`
`(74) Representative: Hiltl, Elmar, Dr. et al
`DIEHL.GLAESER.HILTL & PARTNER,
`Patentanwalte,
`Augustenstrasse 46
`80333 Mflnchen (DE)
`
`(54)
`
`ANTITUMOR AGENTS AND PROCESS FOR PRODUCING THE SAME
`
`Anticancer agents which contain as the active
`(57)
`ingredient heme oxygenase inhibitory metalloporphyrin
`derivatives which are conjugated with amphipathic or
`water-soluble polymers (in particular, Zn-protoporphyrin
`(ZnPP) conjugated with polyethylene glycol). Because
`of being conjugated to amphipathic orwater-soluble pol-
`
`ymers, such as polyethylene glycol, the active ingredi-
`ent can be administered by intravenous injection and
`can exertaremarkable anticancereffect owing to tumor-
`selective delivery.
`
`EP1421938A1
`
`Printed by Jouve, 75001 PARIS (FR)
`
`

`

`EP 1 421 938 A1
`
`Description
`
`Field of the invention:
`
`[0001] The present invention relates to anticancer agents with little side effect and excellent tumor accumulation
`thereby exhibiting very potent anticancer effect, and the preparation method of the same. More precisely, it relates to
`anticancer agents containing as the active ingredient heme oxygenase inhibitory metalloporphyrin derivatives that are
`conjugated with amphipathic or water-soluble polymers. And it relates to also a preparation method of the same with
`high efficiency.
`
`Background technology:
`
`[0002] The inventors of the present invention have investigated the relationship between cancer growth or its sup—
`pression and the activity of heme oxygenase, and found that heme oxygenase is highly expressed in tumor tissues.
`The heme oxygenase degrades heme and produces bilverdin, carbon monoxide and free iron in tumor or normal
`tissues.
`
`[0003] Bilverdin is readily converted into bilirubin in the cells, and this bilirubin is a very potent antioxidant. Thereby,
`bilirubin can be a defense molecule against active oxygen such as peroxide, H202. or nitric oxide etc. that are generated
`by leukocytes of the hosts (cancer patients). Namely, bilirubin, thus generated will nullify the toxic oxidative defense
`power against cancer cells or infecting microbes ofthe host. Therefore, if one blocks heme oxygenase, no bilirubin will
`be available and tumor cells will be killed by the oxidative molecules generated by leukocytes as a result of an innate
`defense state.
`
`[0004] The inventors had tried to see the antitumor effect of zinc protoporphyrin (ZnPP), an inhibitor of heme oxy—
`genase, administered into the tumor feeding artery of tumor bearing rats thereby targeting the inhibitor into the tumor
`loci selectively, and they indeed confirmed antitumor effect in rats (K. Doi et al.: Br. J. Cancer 80, 1945—54, 1999).
`[0005] However, there are several problems to use ZnPP per se as an antitumor agent. First,
`it is almost insoluble
`in water per se, thus, one must use an oily formulation to solubilize ZnPP, and such oily formulated agent may be only
`injectable via the tumor-feeding artery, and this is rather too elaborate and far advanced skill is required for this pro-
`cedure compared with ordinary intravenous or subcutaneous injection. Second, native or original ZnPP has no guar-
`antee for selective accumulation of ZnPP in cancer tissues, and to exert tumor selective anticancer effect, whereas
`the drug will be widely distributed to the whole body besides the tumor. Therefore, unexpected side effects are con-
`cerned.
`
`On the contrary, the inventors are experts in tumor biology, particularly studied the vascular permeability of solid tumor
`tissues, and know that macromolecular therapeutics would permeate more selectively at the tumor tissue by virtue of
`the unique anatomical character and by the effect of multiple vascular permeability factors; and further, those macro—
`molecules are retained in the tumor tissues for long periods.
`Thus, this phenomenon was coined "enhanced permeability and retention (EPR)—effect" (Y. Matsumura, H. Maeda:
`Cancer Res. 47: 6387-92, 1986: H. Maeda: In Advances in Enzyme Regulation (by G. Weber ed), Elsevier Scientific
`Ltd., Amsterdam, 41, 189-207, 2001).
`[0006] According to the EPR-effect, drugs with molecularsize largerthan 40,000 exhibit a high concentration in blood
`plasma for a prolonged time, and several hours to days after intravenous injection, whereas an intratumoral concen-
`tration will result in a multiple time, more precisely in 24-48 hr. This means, making the apparentdrug size greater than
`40,000 would make possible a selective tumor targeting of such macromolecular drugs.
`[0007] Meanwhile, various metal porphyrin derivatives having inhibitory activity against heme oxygenase, and im-
`proved methods of their administrations as a whole were studied. The result is that an amphipathic or water soluble
`polymer conjugation to the metal protporphyrins made it possible to yield water soluble metal porphyrin derivatives
`and they can be administered not only arterially but also intravenously which has more versatile and easy clinical uses.
`They exhibited EPR—effect by polymer conjugation yielding a highly efficient accumulation in tumor, and enzyme inhib—
`itory activity against heme oxygenase is retained for long periods. As a result, only 2 to 3 times of injections made it
`possible to suppress tumor growth completely in mice, which was a remarkable result.
`[0008]
`Previously, metal porphyrin derivatives possessing heme oxygenase inhibitory activity with amphipathic or
`water soluble polymer conjugation were never reported, nor was the method of their preparation before our own. The
`inventors have developed the method for synthesis of amphipathic or water-soluble polymer conjugation of metallo-
`porphyrin via an amide linkage. Resulting polymer conjugated metalloporphyrin derivatives are novel series of com-
`pounds not reported previously.
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`Disclosure of the Invention
`
`EP 1 421 938 A1
`
`[0009] The present invention are anticancer agents containing as the active ingredient metalloporphyrin derivatives
`having inhibitory activity against heme oxygenase, especially Zn-protoporphyrin (ZnPP) conjugated with amphipathic
`polymers which are both water and lipid soluble or water soluble polymers.
`[0010] The present invention is also a series of novel useful compounds for an ingredient of anticancer agents where
`amphipathic or water-soluble polymers and heme oxygenase inhibitory metalloporphyrin derivatives are conjugated
`via amide bonds, and a preparation method of the compounds.
`
`Brief Explanations of drawings
`
`[0011]
`
`Figure 1 shows the gel filtration chromatography of diaminoethane coupled protoporphyrin and PEG-conjugated
`protoporphyrin.
`Figure 2 shows a Lineweaver-Burk plot of PEG-ZnPP inhibitory profile against heme oxygenase.
`Figure 3 shows flow-cytometric analysis data where PEG-ZnPP treated cultured cancer cells exhibit a more oxidant
`exposured profile.
`Figure 4 shows an antitumor effect of PEG-ZnPP in a mouse model with a solid tumor.
`Figure 5 shows the profile of body weight change during or after intravenous administration of PEG-ZnPP.
`
`Most preferable embodiment for carrying out the invention
`
`[0012] Amphipathic or water soluble polymers to be conjugated include polyethylene glycol (PEG), polypropylene
`glycol (PPG), polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), various gelatins, and their derivatives such as suc—
`cinylated forms, polyamino acids (eg. polymerized aspartic acid, glutamic acid, lysine, alanine, glycine, proline, tyrosine,
`etc.), hydroxypropyl and other alkyl acrylate polymers, styrene maleic acid copolymers(SMA), and their derivatives.
`Among these polymers with amphipathic and water-soluble characters, PEG and SMA are more preferable. PEG with
`molecular weight of 2000-5000 is preferably used.
`[0013]
`SMA is a copolymer of styrene and maleic acid in the alternative order where the carboxyl group of maleic
`acid can be utilized to conjugate with metalloporphyrin directly or indirectly. SMA can be used as such or as its deriv-
`atives where maleic acid is partially esterified.
`[0014] Metal porphyrin derivative is a complex porphyrin compound, where the metal is chelated in stable coordina-
`tion to the porphyrin ring, and protoporphyrin is preferably used because of its easy availability among porphyrin com-
`pounds.
`[0015] Among the metals to be coordinated, iron that give no heme oxygenase inhibitory action, mercury with poi—
`sonous nature, monovalent metals which do not form coordinated chelation, can not be used. Although, various metals
`other than above such as zinc, tin, cobalt, and copper can be used. Among them, tin and zinc complexes are more
`preferred. However, tin is also known to be poisonous. Thus, ZnPP is most preferable and its chemical structure is
`shown in formula B.
`
`COOH
`
`COOH
`
`(B)
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`EP 1 421 938 A1
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`[0016] The anticancer agents of the present invention are any macromolecular compounds obtained by conjugation
`of a metal porphyrin with amphipathic or water-soluble polymers. However we found it difficult to carry out the conju-
`gation of the polymers to the metal porphyrin directly, because a metal porphyrin derivative is water-insoluble. To
`undertake this chemical conjugation, it is preferable to conjugate the polymers to porphyrin before the coordination of
`metal, then to coordinate the metal.
`[0017] The conjugation of porphyrin with the polymer can be facilitated directly as well as by introducing a desired
`functional spacer group.
`[0018]
`For example, in the synthesis of ZnPP, the polymer can be directly conjugated to the two carboxyl groups in
`protoporphyrin, but this direct conjugation method is not advantageous because of poor activity of said carboxyl groups
`for this reaction. The inventors of the present invention studied effective synthesis methods of PEG conjugated ZnPP,
`and succeeded to synthesize ZnPP conjugated with PEG via an amide bond (formula A).
`
`(A)
`
`Therein, R means an amphipathic or water-soluble polymer, and Me is a metal.
`[0019] The polymer conjugated ZnPP B may be synthesized by a successive reaction as follows:
`
`(1)|ntroduction of amino group to protoporphyrin IX;
`(2)Conjugation of the polymer, and lastly
`(3)Coordination of Zn into porphyrin ring.
`
`For example, the scheme of synthesis of PEG conjugated ZnPP is shown diagrametrically by stepwise reac-
`[0020]
`tions as follows.
`
`] Protoporphyrin IX (compd.(1)) is activated with ethyl chloroformate in tetrahydrofuran (compd.(2)).
`[Reaction (a
`] Protoporphyrin with diamino group (compd.(3)) can be obtained by addition of ethylene diamine.
`[Reaction (b
`[Reaction (c ] PEG is introduced into protoporphyrin ring by addition of activated PEG (compd.4).
`[Reaction (d ]. Lastly, PEG-ZnPP (compd.5) is obtained by addition of zinc acetate to the reaction product of the
`Reaction (d). One can replace Zn for tin (Sn) and obtain PEG-Sn-PP by addition of tin acetate
`
`vvvv
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`EP 1 421 938 A1
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`EP 1 421 938 A1
`
`[0021] Other compounds than PEG, with amphipathic or water soluble polymers, such as SMA, can be attached to
`protoporphyrin similarly by condensation reaction of compd.(3) and SMA.
`[0022] Heme oxygenase inhibitory methalloprotoporphyrin, that is conjugated with amphipathic orwater-soluble pol-
`ymers shown in formula A, is selectively accumulated in solid tumors and exhibit excellent antitumor activity. Thus it
`is a novel and useful antitumor substance. The compd.(5), which is a typical example of the compound A, wherein
`metal is zinc and R is PEG, was synthesized by the scheme (a) to (d). The chemical structure of the reaction product
`was confirmed by following analysis.
`[0023]
`Firstly; the evidence of the amino group of ethylene diamine that was introduced into protoporphyrin (compd.
`3) is confirmed by (1 )lnfrared spectra with absorbance at 1641cm'1, 1552cm'1 showed a new formation of the amide
`bond in the compound (structure of compd.3). (2)Determination of molecular weight of the compound by mass spec-
`troscopy (MS) showed 646, identical to the value calculated by formula based on the compd.3.
`[0024] Then, PEG (mw about 5000) was coupled to amino group introduced into the protoporphyrin (compd.3), and
`zinc is chelated. The structure of thus obtained ZnPP was identified by determination of the molecular weight and
`absorption spectra (UV/Vis).
`[0025] Determination of the molecular weight showed mass of near 11,000 Da by TOF/MS (time of flight-mass spec-
`troscopy). UV absorption showed a max. peak at 425, 543, and 583 indicating formula (5) to be PEG-ZnPP.
`[0026] The scheme of PEG-ZnPP synthesis using protoporphyrin IX as starting material via reaction steps [a] - [d]
`is a novel manufacturing method. The obtained polymer conjugated metalloporphyrin is readily water-soluble and it
`may be used as injection solution either intravenously or arterially.
`
`[Examples]
`
`[0027] The process for preparing the PEG—ZnPP. inhibitory activity of the PEG—anp towards heme oxygenase and
`anticancer effect of PEG—ZnPP by intravenous injection according to the present invention shall be explained in detail
`with the following examples. However it should be understood, that the present invention shall not limited to these
`examples.
`
`[Example of Manufacturing]: Synthesis of polyethylene glycol conjugated ZnPP (PEG-ZnPP)
`
`100mg of protoporphyrin IX was dissolved in 20 ml of tetrahydrofuran, and 2.45 ml of triethylamine was added
`[0028]
`to this solution. This solution was kept at about 0°C on ice, then 1.7 ml of ethyl chloroformate was added to this dropwise
`understirring, and allowed to react further for two hrs. Subsequently, triethylamine HCI salt being formed was removed
`by filtration, and 1.2 ml ethylene diamine was added, and the reaction was continued at room temperature for 24 hrs.
`The reaction mixture was then subjected to vacuum evaporation to remove tetrahydrofuran, and the solid material
`obtained was washed 7 times with 50ml of distilled water yielding 60 mg of porphyrin derivative having two amino
`groups per molecule (reaction a and b).
`[0029]
`Five mg of compound (3) was dissolved in 25 ml ofchloroform, and 800 mg ofsuccinimidoester of polyethylene
`glycol (Shearwater; PEG, MW5000) was added to this solution, and reacted for24 hrs under stirring at room temperature
`[reaction 0].
`[0030]
`PEG-conjugated protoporpyrin thus obtained was subjected to gel filtration chromatography on Sephadex
`LH60 using chloroform as eluent. The result of the gel filtration chromatography showed that unreacted aminated
`compound (3) did not exist in the preparation of PEG-conjugated protoporpyrin at all. It showed all aminated protopor-
`phyrin reacted with PEG to form polymeric form of protoporphyrin. Unmodified protoporphyrin, if any, was eluted at
`fraction No. 20, where elution volume was similar to aminated protoporphyrin.
`[0031]
`40mg of zinc acetate was added to the PEG-PP solution and allowed for two hrs at room temperature yielding
`PEG-conjugated zinc protoporphyrin (PEG-Zn-PP) (reaction d).
`
`[Experimental Example 1]: Inhibitory activity of PEG—ZnPP against heme oxidase.
`
`[0032] This was examined using purified heme oxygenase fraction derived from rat spleen. It was assayed at 37 °C
`in the presence of hemin, the substrate of heme oxygenase, cofactor (NADPH, nicotine adenine dinucleotide), and
`cytosolic fraction containing bilirubin reductase, in which biliverdin formed by the oxygenase is converted to bilirubin.
`[0033]
`Bilirubin was extracted with chloroform and quantified by absorption at 465 nm. By the addition of either
`PEG-ZnPP, or unmodified ZnPP, or no inhibitor, their effecton heme oxidase was examined, and the Lineweaver-Burk
`plot of heme oxygenase activity was plotted during the inhibition by PEG-ZnPP. The result is shown in Figure 2, indi-
`cating that PEG-ZnPP inhibits the heme oxygenase in a dose dependent manner, and inhibitory constant (Ki) was 0.13
`uM. Mode of inhibition was competitive, and the value was equivalent to that of unmodified ZnPP. (Ki = 0.12 uM)
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`[Experimental Example 2]: Effect of PEG—ZnPP on cultured tumor cells.
`
`EP 1 421 938 A1
`
`Lung adenocarcinoma cell line A549 cells were plated in a plastic dish and after overnight culture, 5 ”M and
`[0034]
`10 uM of PEG-ZnPP dissolved in distilled water were added to the culture dishes. Then, 8 hrs after cultivation at 37
`°C, a reagent that quantifies oxidative stress, called dichIorodihydrofluorescein diacetyl ester (DCDHF), was added
`and followed by cell culture for 30 minutes. Under oxidative stress, this DC DHF will become oxidized and will fluoresce
`due to oxystress generated by formation of fluorescein in cells.
`[0035] Quantification of fluorescence intensity. represents the extent of oxidative stress induced in the cells. Then,
`cultured cells were trypsinized and recovered cells were subjected to the flow cytometrory analysis and fluorescence
`cell population was quantified. The results are shown in Figure 3, where the effect of PEG-Zn PP at 5 uM, 10 uM,
`is
`compared with that of no drug. It is clearfrom these data in the Fig. 3 that PEG—ZnPP brought about a higher intracellular
`oxidative state in the dose dependent manner of PEG—ZnPP.
`
`[Experimental Example 3]: Inhibition of heme oxygenase in solid tumor model in mouse.
`
`In male ddY mice with mean body weight of 35 g, 8180 sarcoma cells were implanted in the dorsal skin, and
`[0036]
`when solid tumor size becomes 5 mm in cross diameter after about one week, PEG-ZnPP dissolved in distilled water
`was injected via the tail vein (i.v.) at 0.5 mg ZnPP equivalent per Kg body weight. The solid tumors were removed after
`24 hr, and heme oxygenase activity was quantified similarly as described in Example 1. A control mouse received
`distilled water without PEG-ZnPP. The tumor specimens were obtained and treated similarly. As shown in Table 1,
`PEG-ZnPP given iv (tail) showed significant reduction of the heme oxygenase activity. Unmodified ZnPP could not
`be administered i.v. because of its difficulty in solubility.
`
`[Table 1]
`
`Inhibition of intratumor heme oxygenase by PEG-ZnPP given via the tail vein.
`
`Drug
`
`Activity of heme oxygenase in tumor tissue.
`(n mol bilirubin/mg protein/hr)
`
`Control, none
`
`
`
`Group of PEG—ZnPP administered
`
`Unmodified ZnPP administered
`
`Impossible to solubilize in water (can not be injected)
`
`[Experiment 4]: Antitumor effect of PEG-ZnPP and change of body weight in mice bearing solid tumor.
`
`Similar to Experiment 3 above, with sarcoma S 180 of mice implanted under the dosal skin of ddY mice, and
`[0037]
`after 10, 13 and 15 days after tumor implantation, PEG-ZnPP at 30 n mole, 30 n mole and 50 n mole (3 times only),
`respectively, was injected into the tail vein respectively (see also arrow marks in Figure 4). Control mice received
`distilled water instead of PEG-ZnPP. The sizes of the tumors were measured every week day as shown in Figure 4.
`It
`is clear that the PEG-ZnPP group showed remarkable suppression of tumor growth compared with control group.
`[0038] The body weight of both treated and non—treated mice was measured simultaneously as seen in Figure 5.
`There was no remarkable body weight loss in the group treated with PEG—ZnPP.
`
`
`Applicability of the Invention in the Industrial Sense
`
`[0039] According to the present invention, metal porphyrin derivatives which are inhibitory against heme oxygenase,
`can be made both water-soluble and lipid soluble. They make the derivatives to become an intravenously injectable
`medicament by conjugation with amphipathic or water-soluble polymers. This is a novel medicament having an excel-
`lent tumor selective accumulation. An effective preparation method of this compound was also found.
`[0040] The anticancer agents according to the present invention have an excellent anticancer effect without gener-
`ating any appreciable side effect or toxicity.
`[0041] Thus the polymer conjugated anticancer agents according to the present invention are highly useful drugs
`having an excellent tumor selective targeting property with a new mode of action different from many of the known low
`molecular weight anticancer drugs.
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`Claims
`
`EP 1 421 938 A1
`
`1. Anticancer agents which contain as the active ingredient heme oxygenase inhibitory metalloporphyrin derivatives
`which are conjugated with amphipathic or water-soluble polymers.
`
`2. The anticancer agents according to Claim 1, wherein the heme oxygenase inhibitory metalloporphyrin derivatives
`are complex compounds in which a metal atom is chelated to protoporphyrin in stable coordination.
`
`3. The anticancer agents according to Claims 1 or 2, wherein the amphipathic or water-soluble polymers are poly-
`ethylene glycol (PEG) or a styrene-maleic acid copolymer (SMA).
`
`4. The anticancer agents according to Claims 1—3, wherein the heme oxygenase inhibitory metalloporphyrin deriva—
`tives are conjugated with amphipathic or water—soluble polymers via amide bonds.
`
`5. The anticancer agents according to Claims 1-4, wherein the metal is zinc.
`
`6. Heme oxygenase inhibitory metalloprotoporphyrin derivatives which are conjugated with amphipathic or water
`soluble polymers represented by the general formula A
`
`(A)
`
`wherein "Fl" means amphipathic or water soluble polymers and "Me" is a metal.
`
`7. A preparation method of heme oxygenase inhibitory metalloporphyrin derivatives, which are conjugated with am-
`phipathic or water soluble polymers according to Claim 6, which comprises introducing ethylene diamine to pro-
`toporphyrin IX, introducing the polyethylene glycol chain into protoporphyrin by adding activated polyethylene gly-
`col, and finally adding a metal salt to introduce the metal atom into the porphyrin ring.
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`EP 1 421 938 A1
`
`Figure 1
`
`EEanBoaumfimacooémm
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`(mu 007) eouquosqV
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`

`

`EP 1 421 938 A1
`
`Figure 2
`
`unmodified Zn PP
`
`
`
`
`
`
`
`1/v(bilimbinnmol/mg/hr).
`
`
`
`
`
`—o.1
`o
`03
`0.2
`0.3
`1/hemin (conc)(pM~1)
`
`no inhibitor
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`EP 1 421 938 A1
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`Figure 3
`
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`160
`
`1
`
`No.ofCells
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`1
`
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`1'
`PEG-ZnPP(1 OpM)
`
`~
`
`101
`
`102
`
`Fluorescence intensity
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`EP 1 421 938 A1
`
`Figure 4
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`EP 1 421 938 A1
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`Figure 5
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`EP 1 421 938 A1
`
` International application No.
`
`INTERNATIONAL SEARCH REPORT
`PCT/JP02/08707
`
` A. CLASSIFICATION OF SUBJECT MATTER
`
`
`
`Int.c17 A61K31/409, 47/48,
`A61P35/00, 43/00, co7D4e7/22
`
`According to Intematicnal Patent Classification (lPC) or to both national classification and IPC
`
`
`Bi FIELDS SEARCHED
`
`Minimum documentation searched (classification system followed by classification symbols)
`
`Int: .Cl7 A61K31/409 , 47/48 , A61P35/00 , 43/00, CO7D487/22
`
`Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
`
`
`
`
`
`Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
`
`
`
`CAPLUS (STN) , CAOLD (STN) , REGISTRY (STN) , WPI/L (DIALOG)
`
`
`
`
`C. DOCUMENTS CONSIDERED TO BE RELEVANT
`
`
`
`
`
`
`
`Relevant to claim No.
`1- 7
`
`
`
`
`
`
`
`
`Citation of document with indication, where appropriate of the relevant passages
`WO 91/18006 A
`(DIATRON CORP. l,
`28 November, 1991 (28.11.91),
`Concerning the compound in which PEG is bonded to
`porphyrin, see the claims, Fig. 1,
`lines 16 to 31
`of paragraph 5,
`lines 14 to 23 of paragraph 6, and
`example 1
`5: EP 529002 Al
`& JP 5-508015 A
`
`5: US 5403928 A
`
`
`
`
`
`
`
`(Hidetoshi TSUCHIDA) ,
`JP 55—144028 A
`10 November, 1980 (10.11.80),
`Concerning the compound in which PEG is bonded to
`protoporphyrin, see the whole specification.
`(Family: none)
`
`
`
`‘X"
`
`“Y”
`
`“L”
`
`"0"
`
`C] See patent family annex.
`CZ] Further documents are listed in the continuation of Box C.
`‘T”
`later document published after the inlemational filing date or
`‘
`Special categories of cited documents:
`priority date and not in conflict with the application but cited to
`“A”
`document defining the general state of the art which is not
`understand the principle or theory underlying the invention
`considered to be ofpartitmlar relevance
`document ofparticular relevance; the claimed invention cannot be
`Il3”
`earlier document but published on or after the international filing
`considered novel or cannot be considered to involve an inventive
`date
`step when the document is taken alone
`document which may throw doubts on priority claim(s) or which is
`document ofparticular relevance; the claimed invention cannot be
`cited to establish the publication date ofanothcr citation or other
`considered to involve an inventive step when the document is
`special reason (as specified)
`combined with one or more other such documents, such
`document referring to an oral disclosure, use, exhibition or other
`combination being obvious to a person skilled in the art
`means
`document member of the same patent family
`“At"
`document published prior to the intemaiional filing date but later
`"P”
`
`
`than the priority date claimed
`
`
`
`Date of the actual completion of the international search
`Date of mailing of the international search report
`
`
`
`20 November, 2002 (20 . 11 . 02)
`
`17 December, 2002 (17 . 12 . 02)
`
`
`
`
`
` Name and mailing address of the ISA/ Authorized officer
`
`Japanese Patent: Off ice
`
`Facsimile No.
` Telephone No.
`
`Form PCT/ISA/ZIO (second sheet) (July 1998)
`
`14
`
`

`

`EP 1 421 938 A1
`
`INTERNATIONAL SEARCH REPORT
`
`
`
` International application No.
`PCT/JP02/08707
`
`C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT
`
`Y
`
`Y
`
`Y
`
`P,X
`
`XY
`
`
`
`
`
`
`
`
`
`
`Citation of document, with indication, where appropriate, of the relevant passages
`
` DE 2645079 A (BAYER ERNST),
`13 April, 1978 (13.04.78),
`
`
`Concernig the compound in which PEG is bonded to
`
`porphyrin, see p.4.
`
`
`(Family: none)
`
`
` K. D01, et a1., “Induction of heme oxygenase-1 by
`
`
`nitric oxide and ischaemia in experimental solid
`
`
`tumors and implications for tumor growth”,
`
`
`
`Br. J. Cancer, 1999, Vol.80, No.12, pages 1945 to
`1954
`Concerning the haem oxygenase inhibiting action
`
`
`and tumor suppression action of a zinc complex of
`protoporphyrin, see the whole article.
`
`(INSTITUT FUR DIAGNOSTIKFORSCHUNG
` US 5849259 A
`
`
`GMBH .
`)
`r
`
`
`15 December, 1998 (15.12.98),
`
`
`Concerning derivatives in which a side chain is
`
`
`introduced into protoporphyrin through ethylene
`
`
`—diamine and their preparing methods, see the
`
`
`compounds of working examples 2,
`10—12 and the
`
`
`Claims.
`
`
`& JP 8—504399 A
`& W0 94/7894 A1
`
`S. K. SAHOO, et a1., Pegylated zinc protoporphyrin
`A water-soluble heme oxygenase inhibitor with
`tumor
`'
`
`
`—targeting capacity, Bioconjugate Chem., 2002,
`
`
`Vol.13, No.5, pages 1031 to 1038
`
`
`Chemical Abstracts, 1995, Vol.124, abstract No.
`
`
`219685 & Su BINGYIN, et a1., Synthesis of water
`
`
`
`
`soluble derivatives of porphyrin metal complex and
`
`
`its effect on tumor cell growth, Zhongguo Yaoxue
`
`
`
`Zazhi
`(Beijing), 1995, Vol.30, No.12, pages 746 to
`748
`
`
`
`Form PCT/lSA/ZlO (continuation of second sheet) (July 1998)
`
`15
`
`